Enterohemorrhagic E. coli (EHEC) O157:H7 causes bloody diarrhea and hemolytic uremic system (HUS) throughout the world. EHEC has a very low infectious dose, making it difficult to control epidemiologically. EHEC colonizes the large intestine where it causes attaching and effacing (AE) lesions and also produces Shiga toxins that are responsible for the major symptoms of HUS. Each year, EHEC causes more than 70,000 illnesses, 2000 hospitalizations, and 70,000 deaths in the United States alone. EHEC senses a bacterial autoinducer (AI-3) produced by the normal gastrointestinal flora, as well as the hormones epinephrine and norepinephrine produced by the host to activate expression of its virulence genes. Additional regulation of virulence genes occurs through the transcription factor QseA and small RNA molecules (sRNAs). However, many aspects concerning QseA and sRNA gene regulation, which are critical for virulence, remain poorly understood. This application represents a comprehensive effort to explore the functions of QseA and of sRNAs responsible for virulence gene regulation in EHEC. The Specific Aims are (1) To determine the mechanism of QseA activation and repression of target genes, (2) To characterize the role of QseA in EHEC virulence gene regulation, and (3) To elucidate the roles of small RNA (sRNA) molecules in post-transcriptional gene regulation in EHEC. The proposed experimental approaches will achieve a better understanding of the mechanisms employed by EHEC to activate its virulence genes and may reveal potentially novel aspects of EHEC pathogenesis. These data may ultimately lead to the development of unique treatment strategies for EHEC infection.